Human respiratory syncytial virus (RSV) is the most important pediatric respiratory pathogen worldwide. This ubiquitous, highly infectious agent emerges each year in seasonal epidemics. Nearly everyone is infected at least once within the first two years of life. RSV disease is responsible for considerable morbidity and mortality, especially in the very young; in the United States it causes an estimated 91,000 hospitalizations and 4500 deaths annually, and its impact is much greater in less affluent countries. RSV also has come to be recognized as an important agent of disease of immunocompromised adults and of the elderly.
Resistance to RSV reinfection induced by natural infection is incomplete but increases incrementally with repeated exposure. Thus, RSV can infect multiple times during childhood and life, but serious disease usually is limited to the first and sometimes second infections of life. The minimum goal of RSV immunoprophylaxis is to induce sufficient resistance to prevent serious disease associated with the initial infections.
A number of attenuated RSV strains were developed and evaluated as vaccines during the 1960's and 70's, but they were found to be either over- or under-attenuated, and in some cases exhibited genetic instability, as is common for single-stranded RNA viruses. Current strategies under investigation for RSV vaccine development are principally the parenteral administration of purified viral antigen or the development of live attenuated RSV for intranasal administration. The intranasal route provides direct stimulation of local immunity. It also partially abrogates the immunosuppressive effects of RSV-specific maternally derived serum antibodies, which typically are found in the very young. The parenteral administration of inactivated RSV or purified RSV antigen in experimental animals appears to be associated with enhanced immunopathology upon subsequent virus challenge, similar to the enhanced RSV disease associated with a formalin-inactivated vaccine evaluated in the 1960's. But this effect has never been observed with RSV infection of the respiratory tract, suggesting that live attenuated viruses have an important advantage in safety. To date, however, there is no approved vaccine or highly effective antiviral therapy for RSV.
Research efforts to produce a suitable RSV vaccine are impeded by poor viral growth in tissue culture, a lengthy replication cycle, virion instability, a negative-sense RNA genome, and a complex genome organization and gene products. RSV is a member of the pneumovirus genus of the paramyxovirus family, and its genome of single-stranded negative-sense RNA of 15,222 nucleotides has been sequenced completely for wild-type strain A2 virus as well as for an attenuated derivative thereof.
Some aspects of RNA synthesis by RSV appear to follow the general pattern of nonsegmented negative strand viruses. The genome template is tightly encapsidated with the major nucleocapsid (N) protein and is associated with the phosphoprotein (P) and large (L) polymerase subunit protein. Transcription begins at the 3' extragenic leader region and proceeds along the entire length by a sequential, stop-start mechanism guided by short template signals flanking the genes. This yields at least ten major species of mRNA which encode at least ten major proteins. RNA replication occurs by a switch to the synthesis of a full length positive-sense "antigenome" which also is tightly encapsidated and serves as the template for the synthesis of progeny genome.
The viral genomic RNA of negative-strand viruses is not infectious alone as free RNA. In virions or intracellularly, viral RNA is always found tightly encapsidated in a ribonucleoprotein core. This nucleocapsid contains the viral proteins necessary for transcription and replication and has long been regarded as the minimum unit of infectivity (Brown et al., J. Virol. 1: 368-373 (1967)). Thus, it has been recognized that the generation of biologically active synthetic viral RNA from cDNA will require complementation by viral protein, leading to the assembly of functional nucleocapsids (Collins et al., Proc. Natl. Acad. Sci. USA 88: 9663-9667 (1991), and Collins et al., Virology 195: 252-256 (1993)). The ability to produce live RSV from cDNA is of particular importance because it would permit the introduction of specific engineered changes, including attenuating mutations, into the genome of infectious virus in an effort to produce safe and effective RSV vaccines.
Short, internally-deleted analogs of genome or antigenome RNA ("minigenomes") have been shown to participate in transcription and replication when synthesized intracellularly in the presence of the appropriate viral proteins. For two rhabdoviruses, rabies and vesicular stomatitis viruses, infectious virus has been produced by coexpression of a complete cDNA-encoded antigenome RNA in the presence of the N, P and L proteins (Schnell et al., EMBO J. 13: 4195-4203 (1994) and Lawson et al., Proc. Natl. Acad. Sci. USA 92: 4477-4481 (1995)).
RSV possesses a number of properties which distinguishes it and other members of the genus Pneumovirus from the better characterized paramyxoviruses of the genera Paramyxovirus, Rubulavirus and Morbillivirus. These differences include a greater number of mRNAs, an unusual gene order at the 3' end of the genome, species-to-species variability in the order of the glycoprotein and M2 genes, a greater diversity in intergenic regions, an attachment protein that exhibits mucin-like characteristics, extensive strain-to-strain sequence diversity, and several proteins not found in any or most of the other nonsegmented negative strand RNA viruses.
RSV remains the most common cause of severe viral lower respiratory tract disease in infants and children. Consequently, an urgent need remains for the ability to engineer a safe and effective vaccine that is able to prevent the serious illness in this population that often requires hospitalization. Quite surprisingly, the present invention fulfills this and other related needs by providing methods for introducing defined, predetermined changes into infectious RSV.